SJOBERG ERIC (US)
| CLAIMS 1. An antibody, wherein the antibody binds a sialic acid binding-immunoglobulin type lectin (siglec) and wherein the antibody comprises at least one human antibody variable domain. 2. The antibody of claim 1, wherein the siglec comprises siglec12. 3. The antibody of claim 1 or 2 comprising at least one complementarity determining region (CDR) amino acid sequence selected from Table 5. 4. The antibody of claim 3 comprising at least one variable domain comprising a CDR amino acid sequence set selected from Table 6. 5. The antibody of claim 4 comprising a pair of variable domains comprising a CDR amino acid sequence set pair selected from Table 7. 6. The antibody of any one of claims 3-5 comprising at least one antibody framework (FR) amino acid sequence selected from Table 8. 7. The antibody of any one of claims 1-6 comprising a variable domain amino acid sequence selected from Table 2 or encoded by a nucleic acid sequence selected from Table 3. 8. The antibody of claim 7 comprising a variable domain amino acid sequence pair selected from Table 4. 9. The antibody of any one of claims 1-8, wherein the antibody binds to the siglec12 extracellular domain. 10. The antibody of claim 9, wherein the antibody binds to the siglec12 long isoform extracellular domain, the siglec12 short isoform extracellular domain, a subdomain of the siglec12 long isoform extracellular domain, and/or a subdomain of the siglec12 short isoform extracellular domain. 11. The antibody of claim 10, wherein the subdomain of the siglec12 long isoform extracellular domain and/or the subdomain of the siglec12 short isoform extracellular domain comprises the Vset1 (S12-V1) region, the Vset2 (S12-V2) region, the C2set1 (S12-C1) region, or the C2set2 (S12-C2) region. 12. The antibody of any one of claims 9-11, wherein the antibody binds to a combination of subdomains of the siglec12 extracellular domain. 13. The antibody of claim 12, wherein the combination of subdomains comprises S12-V2 and S12-C1 (S12-V2C1) or S12-V2, S12-C1, and S12-C2 (S12-V2C1C2). 14. The antibody of claim 13, wherein the antibody does not bind to S12-V1 or S12-C2. 15. The antibody of any one of claims 1-14, wherein the antibody does not bind to siglec7 or siglec9. 16. The antibody of claim 15, wherein the antibody does not bind to any siglec proteins other than siglec12. 17. The antibody of any one of claims 1-16, wherein the siglec is associated with a cell. 18. The antibody of claim 17, wherein the cell is a cancer cell. 19. The antibody of claim 18, where the cancer cell is from a cancer selected from any of those listed in Table 10. 20. The antibody of claim 18, wherein the cancer cell is a leukemia cell. 21. The antibody of claim 20, wherein the leukemia cell is an acute myeloid leukemia (AML) cell or a chronic myeloid leukemia (CML) cell. 22. The antibody of any one of claims 17-21, wherein antibody binding to cell-associated siglec induces cell death. 23. The antibody of claim 22, wherein antibody binding to cell-associated siglec inhibits tumor immunosuppression. 24. The antibody of claim 22, wherein antibody binding to cell-associated siglec induces antibody-dependent cellular cytotoxicity (ADCC). 25. The antibody of claim 22, wherein antibody binding to cell-associated siglec induces complement-dependent cytotoxicity (CDC). 26. The antibody of claim 22, wherein the antibody is a bispecific antibody. 27. The antibody of claim 26, wherein the antibody comprises a CD3 binding domain. 28. The antibody of claim 22, wherein the antibody comprises a chimeric antigen receptor. 29. The antibody of any one of claims 1-23, 26, and 27, wherein the antibody comprises a conjugate. 30. The antibody of claim 29, wherein the conjugate comprises a therapeutic agent. 31. The antibody of claim 30, wherein the therapeutic agent comprises a toxin. 32. The antibody of claim 31, wherein the toxin comprises a calicheamicin. 33. The antibody of claim 31, wherein the toxin comprises MYLOTARG®. 34. The antibody of claim 29, wherein the conjugate comprises a detectable label. 35. A method of treating a therapeutic indication in a subject, the method comprising the use of the antibody of any one of claims 1-34. 36. The method of claim 35, wherein the therapeutic indication comprises a cancer-related indication. 37. The method of claim 36, wherein the cancer-related indication comprises leukemia. 38. The method of claim 37, wherein the leukemia comprises AML and/or CML. 39. The method of claim 38, wherein the AML and/or CML include cancer cells expressing siglec12. 40. The method of any one of claims 35-39, wherein the antibody is administered to the subject. 41. The method of claim 40, wherein the antibody is administered to the subject by injection or infusion. 42. A method of detecting a siglec in a subject or a subject sample, the method comprising contacting the subject or subject sample with the antibody of any one of claims 1-21, 29, and 34. 43. The method of claim 42, wherein the antibody comprises a detectable label. 44. The method of claim 42 or 43, wherein the antibody binds to the siglec in the subject or subject sample and wherein a detection reagent is used to detect the bound antibody. 45. The method of any one of claims 42-44, wherein the method comprises detecting the siglec in a subject sample, wherein the subject sample comprises a cell. 46. The method of claim 45, wherein the siglec is detected in the subject sample by fluorescence-associated cell sorting (FACS) analysis. 47. The method of claim 45, wherein the siglec is detected in the subject sample by immunohistochemistry. 48. The method of claim 47, wherein the immunohistochemistry involves a colorimetric- based system or an immunofluorescence-based system for siglec detection. 49. A method of stratifying subjects based on detection of a siglec in the subject or a subject sample, the method comprising: detecting a siglec in the subject or the subject sample according to the method of any one of claims 42-48; and classifying the subject according to the type and/or level of siglec detected. 50. The method of claim 49, wherein the subject is classified according to the presence or absence of siglec12 and/or level of siglec12 in the subject or the subject sample. 51. The method of claim 50, wherein the subject is further classified according to the presence or absence of a specific siglec12 extracellular subdomain and/or level of the specific siglec12 extracellular subdomain in the subject or the subject sample. 52. A method of treating a subject with cancer, the method comprising administering an anti- siglec12 antibody to the subject, the anti-siglec12 antibody comprising: a heavy chain variable domain (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7; and a light chain variable domain (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14. 53. The method of claim 52, wherein the anti-siglec12 antibody binds a subject cancer cell. 54. The method of claim 53, wherein the subject cancer cell is an AML cancer cell. 55. The method of claim 53 or 54, wherein the anti-siglec12 antibody is internalized by the subject cancer cell. 56. The method of claim 55, wherein the anti-siglec12 antibody is conjugated to a cytotoxin. 57. The method of claim 56, wherein the cytotoxin comprises calicheamicin. 58. The method of claim 56, wherein the cytotoxin comprises MYLOTARG®. 59. The method of any one of claims 52-58, wherein the VH comprises the amino acid sequence of SEQ ID NO: 4 and the VL comprises the amino acid sequence of SEQ ID NO: 11. 60. The method of claim 59, wherein the anti-siglec12 antibody comprises a format selected from the group consisting of a Fab format and a whole antibody format. 61. The method of claim 59 or 60, wherein the equilibrium dissociation constant (Kd) for binding of the anti-siglec12 antibody to siglec12 is from about 1 nM to about 10 nM. |
Anti-siglec antibody variable domains of the present disclosure may be encoded by nucleic acid sequences listed in Table 3. In some embodiments, nucleic acid sequences encoding anti-siglec antibody variable domains of the present disclosure may include fragments or variants of the nucleic acid sequences listed. Such fragments or variants may include from about 50% to about 99.9% sequence identity (e.g. from about 50% to about 60%, from about 55% to about 65%, from about 60% to about 70%, from about 65% to about 75%, from about 70% to about 80%, from about 75% to about 85%, from about 80% to about 90%, from about 85% to about 95%, from about 90% to about 99.9%, from about 95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7% or about 99.8%) with one or more of the nucleic acid sequences listed. In some embodiments, nucleic acid sequences encoding anti-siglec antibody variable domains of the present disclosure include codon-optimized variants of the nucleic acid sequences listed. Table 3. Variable domain nucleic acid sequences
In some embodiments, anti-siglec antibodies of the present disclosure include VH and VL domain pairs, wherein each member of the pair includes a variable domain amino acid sequence presented herein and/or is encoded by a variable domain nucleic acid sequence presented herein. In some embodiments, anti-siglec antibodies of the present disclosure include a variable domain pair according to any of those listed in Table 4. Table 4. Variable domain pairs In some embodiments, anti-siglec antibodies of the present disclosure include one or more CDRs with amino acid sequences derived from one or more variable domain amino acid sequence provided in Table 2. In some embodiments, anti-siglec antibodies of the present disclosure include one or more CDRs encoded by nucleic acid sequences derived from one or more variable domain nucleic acid sequences provided in Table 3. Anti-siglec antibody CDRs may include one or more amino acid residues involved in antigen binding (e.g., as determined by co-crystallography with bound antigen). Anti-siglec antibodies of the present disclosure may include CDRs identified through CDR analysis of variable domain sequences presented herein via co-crystallography with bound antigen; by computational assessments based on comparisons with other antibodies (e.g., see Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA.2012. Ch.3, p47-54); or Kabat, Chothia, Al-Lazikani, Lefranc, or Honegger numbering schemes, as described previously. In some embodiments, anti-siglec antibody CDR amino acid sequences may include any of those presented in Table 5, or fragments thereof. In some embodiments, anti- siglec antibodies of the present disclosure include CDRs that include amino acid sequence variants of those listed. Amino acid fragments or variants included in anti-siglec antibody CDRs may include from about 50% to about 99.9% sequence identity (e.g. from about 50% to about 60%, from about 55% to about 65%, from about 60% to about 70%, from about 65% to about 75%, from about 70% to about 80%, from about 75% to about 85%, from about 80% to about 90%, from about 85% to about 95%, from about 90% to about 99.9%, from about 95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7% or about 99.8%) with one or more of the amino acid sequences listed. Table 5. CDR amino acid sequences
In some embodiments, anti-siglec antibodies of the present disclosure include variable domains with a set of CDRs, wherein each member of the set includes a CDR amino acid sequence presented herein. In some embodiments, variable domain CDR amino acid sequence sets may include any of those presented in Table 6. Table 6. Variable domain CDR amino acid sequence sets
In some embodiments, anti-siglec antibodies of the present disclosure include pairs of variable domain CDR amino acid sequence sets presented herein. In some embodiments, anti-siglec antibodies of the present disclosure include variable domain CDR amino acid sequence set pairs presented in Table 7. Table 7. Variable domain CDR amino acid sequence set pairs In some embodiments, anti-siglec antibodies of the present disclosure include one or more framework regions (FRs). FRs may include amino acid sequences derived from variable domain amino acid sequences provided in Table 2. FRs may be encoded by nucleic acid sequences derived from one or more variable domain nucleic acid sequences provided in Table 3. In some embodiments, anti-siglec antibody FRs may include amino acid sequences according to any of those presented in Table 8, or fragments thereof. In some embodiments, anti-siglec antibodies of the present disclosure include FRs that include amino acid sequence variants of those listed. Amino acid fragments or variants included in anti-siglec antibody FRs may include from about 50% to about 99.9% sequence identity (e.g. from about 50% to about 60%, from about 55% to about 65%, from about 60% to about 70%, from about 65% to about 75%, from about 70% to about 80%, from about 75% to about 85%, from about 80% to about 90%, from about 85% to about 95%, from about 90% to about 99.9%, from about 95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7% or about 99.8%) with one or more of the amino acid sequences listed. Table 8. FR amino acid sequences
Anti-siglec antibodies according to the present disclosure may be prepared using any of the antibody sequences (e.g., variable domain amino acid sequences, variable domain amino acid sequence pairs, CDR amino acid sequences, variable domain CDR amino acid sequence sets, variable domain CDR amino acid sequence set pairs, and/or framework region amino acid sequences) presented herein, any may be prepared, for example, as monoclonal antibodies, multispecific antibodies, chimeric antibodies, antibody mimetics, scFvs, or antibody fragments. In some embodiments, anti-siglec antibodies using any of the antibody sequences presented herein may be prepared as IgA, IgD, IgE, IgG, or IgM antibodies. When prepared as mouse IgG antibodies, anti-siglec antibodies may be prepared as IgG1, IgG2a, IgG2b, or IgG3 isotypes. When prepared as human IgG antibodies, anti-siglec antibodies may be prepared as IgG1, IgG2, IgG3, or IgG4 isotypes. Anti-siglec antibodies prepared as human or humanized antibodies may include one or more human constant domains. Siglec protein antigens In some embodiments, anti-siglec antibodies bind to siglec12 protein antigens. In some embodiments siglec12 protein antigens include any of those listed in Table 9. Some siglec12 proteins antigens may be post-translationally processed or recombinantly expressed to exclude leader sequences (underlined in the Table). In some embodiments, siglec12 proteins may include variants or fragments of the sequences listed. Table 9. Siglec12 protein antigen sequences In some embodiments, anti-siglec antibodies of the present disclosure bind to siglec12 protein epitopes on siglec12 protein antigens described herein. Such siglec12 protein epitopes may include or be included within a siglec12 protein antigen amino acid sequence listed in Table 9. In some embodiments, anti-siglec antibodies of the present disclosure bind to siglec12 protein epitopes that include a region formed by a complex of a siglec12 protein epitope with another protein or epitope. In some embodiments, siglec12 protein antigens may be prepared as fusion proteins. Siglec12 protein antigens may be prepared as fusion proteins with antibody Fc regions. Fusions with antibody Fc regions may be prepared to facilitate immunization and generation of siglec12 protein antigen-specific immune response. In some embodiments, siglect12 protein antigens are prepared as fusion proteins with mouse Fc regions. Such Fc regions may include Fc2a fragment (UniProtKB PO1863.1, amino acids 98-330, PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQI SWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKT ELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR TPGK, SEQ ID NO: 177) or may include mouse 2cFc (EPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSE DDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKV NNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWT SNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTT KTISRSLGK, SEQ ID NO: 178). Mouse Fc regions may be used in place of human Fc when carrying out immunizations in mice to minimize generation of antibodies against Fc regions. II. Methods In some embodiments, the present disclosure provides methods of using and evaluating anti-siglec antibodies for therapeutic and diagnostic applications. Therapeutic applications In some embodiments, the present disclosure provides methods of treating therapeutic indications using anti-siglec antibodies (e.g., any of those described herein). The term “therapeutic indication,” as used herein, refers to any disease, symptom, condition, or disorder that may be alleviated, stabilized, improved, cured, or otherwise addressed by some form of treatment or other therapeutic intervention. In some embodiments, the present disclosure provides methods of treating therapeutic indications in subjects by providing antibodies disclosed herein. As used herein the terms “treat,” “treatment,” and the like, refer to any actions taken to offer relief from or alleviation of pathological processes. As it relates to any of the therapeutic indications recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such indications, or to slow or reverse the progression or anticipated progression of such indications. In some embodiments, the present disclosure provides methods of treating therapeutic indications in subjects using anti-siglec antibodies (e.g., any of the antibodies described herein). Such antibodies may include human variable domains. Therapeutic indications may include cancer-related indications, e.g., leukemia, e.g., AML and/or CML. The methods of treatment may include administering anti-siglec antibodies to subjects, e.g., by injection or infusion. Cancer-related indications In some embodiments, therapeutic indications include cancer-related indications. The term “cancer” refers to a collection of diseases characterized by dysfunctional cell growth and division, in some cases spreading between bodily regions. As used herein, the term “cancer-related indication” refers to any disease, disorder, or condition pertaining to cancer, cancer treatment, or pre-cancerous conditions. Cancer-related indications include, but are not limited to, pathological conditions characterized by malignant neoplastic growths, tumors, and/or hematological malignancies. In some embodiments, methods of the present disclosure include treatment of cancer-related indications with anti-siglec antibodies (e.g., any of those described herein). Cancer-related indications include leukemias, which are cancers of the blood or blood forming tissues (e.g., bone marrow and lymphatic system). Leukemias are typically classified by progression and affected cell type. Acute leukemias develop quickly while chronic leukemias develop slowly with worsening effects over time. Lymphocytic leukemias affect lymphocytes, typically due to mutations in cells that make lymphocytes. Myelogenous leukemias involve myeloid cells responsible for blood cell production. In some embodiments, methods of the present disclosure include treatment of acute myeloid leukemia (AML) and/or chronic myeloid leukemia (CML) with anti-siglec antibodies (e.g., any of those described herein). AML involves rapid abnormal myeloblast accumulation, typically in the bone marrow. CML involves mutation in immature myeloid cells leading to a more gradual accumulation of leukemia cells over time that can spread to other parts of the body. Additional cancer-related indications include, but are not limited to, any of those listed in Table 10 below. Anti-siglec antibodies used according to cancer-related indication treatment methods described herein may bind to siglec12 associated with cancer cells, including cancer cells associated with any of the cancer-related indications listed in Table 10. Table 10. Cancer-related indications Anti-siglec antibodies used according to cancer-related indication treatment methods described herein may bind to siglec12 associated with solid epithelial tumors. Such tumors have been shown by immunohistochemical analysis to express siglec12 (United States Publication Number US2020/0003779). According to some methods, siglec12-associated cancer cells targeted according to treatment methods of the present disclosure include hematopoietic cells and/or lymphoid cells. In some embodiments, methods of the present disclosure include treatment of AML and/or CML in subjects by providing anti-siglec antibodies (e.g., any of the anti-siglec antibodies described herein). Siglec-3 or CD33 is expressed on AML cells, including AML blasts in AML patients. Gemtuzumab Ozogamicin (GO, MYLOTARG®, Pfizer, New York, NY) is an antibody drug conjugate that targets CD33 and includes a calicheamicin warhead. GO is the only currently listed form of immunotherapy for AML. In some embodiments, anti-siglec antibodies of the present disclosure target siglec12 on AML and/or CML cells in subjects treated according to methods described herein. In some embodiments, the present disclosure provides methods of treating subjects with cancer by administering an anti-siglec12 antibodies (e.g., any of those described herein). The anti-siglec12 antibodies may include VH amino acid sequences according to any of those presented in Table 2 and/or may be encoded by any of the nucleic acid sequences presented in Table 3. The anti-siglec12 antibodies may include VL amino acid sequences according to any of those presented in Table 2 and/or may be encoded by any of the nucleic acid sequences presented in Table 3. The anti-siglec12 antibodies may bind subject cancer cells. Such cancer cells may include AML cancer cells. In some embodiments, the anti-siglec12 antibodies may be internalized by bound cells. Such antibodies may be ADCs. Therapeutic agents associated with such ADCs may include cytotoxins. Such toxins may include, but are not limited to, radioisotopes, saporins, taxanes, vinca alkaloids, anthracyclines, calicheamicins, duocarmycins, pyrrolobenzodiazepine dimers, and platinum-based agents. Calicheamicins may include MYLOTARG®. In some embodiments, the anti-siglec12 antibodies include a VH with the amino acid sequence of SEQ ID NO: 4 and a VL with the amino acid sequence of SEQ ID NO: 11. The anti-siglec12 antibodies may be whole antibodies or may be antibody fragments (e.g., Fab fragments). The anti-siglec12 antibodies may have an equilibrium dissociation constant (Kd) for binding to siglec12 of from about 0.001 nM to about 10,000 nM (e.g., from about 0.001 nM to about 0.1 nM, from about 0.01 nM to about 1 nM, from about 0.5 nM to about 5 nM, from about 1 nM to about 10 nM, from about 2 nM to about 20 nM, from about 5 nM to about 50 nM, from about 15 nM to about 100 nM, from about 25 nM to about 250 nM, from about 100 nM to about 1,000 nM, or from about 500 nM to about 10,000 nM). Diagnostic applications In some embodiments, the present disclosure provides diagnostic methods involving use of anti-siglec antibodies. Such methods may include detecting siglecs using anti-siglec antibodies (e.g., any of the antibodies described herein). Such methods may include contacting subjects or subject samples with anti-siglec antibodies (e.g., any of those described herein). The anti-siglec antibodies may bind siglec12. The anti-siglec antibodies may include human variable domains. Antibodies used for detection methods may include a detectable label. Detection methods may include the use of detection reagents to detect bound antibodies. As used herein, the term “detection reagent” refers to any compound or substance used to visualize or otherwise observe an object (e.g., a bound antibody or detectable label) or event. Detection reagents may include secondary antibodies or other high affinity compounds (e.g., biotin or avidin) that bind to antibodies being detected or associated conjugates. Detection reagents may be or include substrates for detection of enzymatic detectable labels (e.g., associated with a primary or secondary antibody). Diagnostic applications of the present disclosure may include detecting siglecs in subject samples that include cells. In some embodiment cell-associated siglecs may be detected. Cell-associated siglecs may be detected in subject samples by fluorescence- associated cell sorting (FACS) analysis. In some embodiments, siglecs may be detected in subject samples by immunohistochemistry. Such methods may include the use of colorimetric-based systems or immunofluorescence-based systems for siglec detection. In some embodiments, the present disclosure provides methods of stratifying subjects based on detection of siglecs in subjects or subject samples. Such methods may include detecting siglecs in subjects or subject samples according to any of the methods described herein (e.g., using anti-siglec antibodies) and classifying subjects according to type and/or level of siglecs detected. In some embodiments, subjects may be classified according to the presence or absence of siglec12 and/or level of siglec12 in subjects or subject samples. Subjects may be further classified according to the presence or absence of specific siglec12 extracellular subdomains and/or levels of specific siglec12 extracellular subdomains in subjects or subject samples. Classifications used in subject stratification may include, but are not limited to, classifications by disease type, disease prognosis or severity, suitability for treatment, and type of treatment most likely to be successful or appropriate. III. Definitions Associated with: As used herein, the terms “associated with,” “conjugated,” “linked,” “attached,” and “tethered,” when used with respect to two or more entities, means that the entities are physically associated or connected with one another, either directly or via one or more moieties that serve as linking agents, to form a structure that is sufficiently stable so that the entities remain physically associated, e.g., under working conditions, e.g., under physiological conditions. An “association” need not be through covalent chemical bonding and may include other forms of association or bonding sufficiently stable such that the “associated” entities remain physically associated, e.g., ionic or hydrogen bonding or a hybridization based connectivity. Epitope: As used herein, an “epitope” refers to a surface or region on one or more entities that is capable of interacting with an antibody or other binding biomolecule. For example, a protein epitope may contain one or more amino acids and/or post-translational modifications (e.g., phosphorylated residues) which interact with an antibody. In some embodiments, an epitope may be a “conformational epitope,” which refers to an epitope involving a specific three-dimensional arrangement of the entity(ies) having or forming the epitope. For example, conformational epitopes of proteins may include combinations of amino acids and/or post-translational modifications from folded, non-linear stretches of amino acid chains. Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5ƍ cap formation, and/or 3ƍ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)). Sample: As used herein, the term “sample” or “biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, serum, plasma, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, and urine). Samples may further include a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, and organs. Samples may further refer to a medium, such as a nutrient broth or gel, which may contain cellular components or other biological materials, such as proteins (e.g., antibodies) or nucleic acid molecules. Subject: As used herein, the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants. A subject receiving, requiring, eligible for, or seeking medical treatment is referred to herein as a “patient.” Target: As used herein, the term “target” refers to an entity of interest or attention, which may include a subject, an organ, a tissue, a cell, a protein, a nucleic acid, biomolecule, or a group, complex, or portion of any of the foregoing. In some embodiments, a target may be a protein or epitope thereof for which an antibody has affinity or for which an antibody is desired, designed, or developed to have affinity for. As used herein, the term “target” may also be used to refer to an activity of an agent that is directed to a particular object. For example, an antibody that has affinity for a specific protein “X” may be said to target protein X or may be referred to as an antibody targeting protein X or referred to as a protein X- targeting antibody. Similarly, an object that is the subject of an agent’s activity may be referred to as a “targeted” object. For example, where an antibody has affinity for a specific protein “X,” protein X may be referred to as being targeted by the antibody. IV. Equivalents and scope While various embodiments of the invention have been particularly shown and described in the present disclosure, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the embodiments disclosed herein and set forth in the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process. It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting of” and “or including” are thus also encompassed and disclosed. Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiments of compositions disclosed herein can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art. All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control. Section and table headings are not intended to be limiting. EXAMPLES Example 1. is a novel target for acute myeloid leukemia Siglec12 was reported to be detected in solid epithelial tumors using immunohistochemistry (United States Publication Number US 2020/0003779). However, immunohistochemistry is a non-quantitative methodology and understanding relative siglec12 expression levels between tumor and normal tissue is critical to understanding which cancers will be treatable using a siglec12-targeted immunotherapeutic approach. mRNA levels of a specific target in tissue is an accepted measure of relative expression levels of a specific target. RNAseq data (mRNA levels quantified via next gen sequencing, NGS) from various public databases (TCGA project, Depmap Portal, CCLE, Blueprint Project, and GTEx) were analyzed to discover which cancers make the most suitable targets for siglec12-targeted immunotherapy. Results presented in Fig.1 show differential expression levels of siglec12 between normal tissue and tumors originating in the same tissue (based in whole or in part on data generated by the TCGA Research Network). These results are summarized in Table 11 and reveal that certain solid tumors are preferable cancer targets for siglec12-targeted immunotherapy than others, namely: cervical squamous cell carcinoma and endocervical adenocarcinoma; head and neck squamous cell carcinoma; kidney renal clear cell carcinoma; esophageal carcinoma and stomach and esophageal cancer. Table 11. Increased siglec12 mRNA expression in different solid tumors Further database analysis [DepMap Portal, Broad (2020): DepMap 20Q2 Public, v4] revealed that when measuring mRNA levels (RNAseq) across 1304 different tumor cell lines, siglec12 was most highly expressed in the leukemia cell lines, as shown in Fig.2. In cancer cell lines of hematopoietic and lymphoid tissue origin, both AML (acute myeloid leukemia) and CML (chronic myeloid leukemia) cell lines had significantly greater siglec12 mRNA expression than the other cell lines [based on 2020 Broad Institute CCLE (Cancer Cell Line Encyclopedia) mRNA expression (RNAseq) data, see Fig.3]. Between AML and CML, median siglec12 mRNA expression in AML was 6-fold greater than median levels in CML. CD33 is known to be highly expressed on AML cell lines and AML blasts in AML patients. The CD33-targeted Gemtuzumab Ozogamicin (GO, MYLOTARG®, Pfizer, New York, NY) is an antibody drug conjugate (ADC) that uses a calicheamicin warhead. GO is the only currently listed form of immunotherapy for AML. There are several other AML targets known to be expressed on AML cell lines and blasts in patients that are currently being investigated in clinical and preclinical studies as potential immunotherapeutic targets. Further RNAseq database analysis demonstrated that siglec12 expression levels in AML cell lines compared quite favorably to mRNA levels of other AML targets [based on 2020 Broad Institute CCLE (Cancer Cell Line Encyclopedia) mRNA expression (RNAseq) data, see Fig. 4]. CD33 expression levels on AML cell lines were comparable to those of siglec12, supporting designation of siglec12 as a possible target for immunotherapeutic development and treatment of AML. While AML target over expression on AML blasts is important when evaluating potential immunotherapeutic targets, expression on normal tissues and hematopoietic cells can lead to toxicity and dose-limiting requirements. GO has on-target hematological toxicities associated with CD33 expression on hematopoietic progenitor cells that manifests as persistent thrombocytopenia and prolonged neutropenia (Castaigne, S. et al. Lancet.2012, 379(9825):1508–16). Fig.5 shows mRNA expression levels of siglec12 and other AML target proteins in common hematopoietic cells based on analysis of public mRNA expression data (Blueprint Consortium). Siglec12 is mainly expressed, albeit at relatively low levels, in classical monocytes, macrophage and alternatively activated macrophage. Fig.6 shows expression levels of siglec12 and other AML target proteins in hematopoietic progenitor and stem cells based on analysis of public mRNA expression data (Blueprint Consortium). These cell types are rare, but critical with respect to common hematopoietic toxicities. A significant difference was observed between siglec12 and other AML targets with siglec12 having exceptionally low, or no detectable expression in these hematopoietic stem and progenitor cells. The combination of high over expression of siglec12 on AML cells and lack of expression on hematopoietic stem and progenitor cells supports designation of siglec12 as an exemplary target for AML immunotherapeutic development. Siglec12 and CD33 mRNA expression was compared in normal tissues based on analysis of public data (The Genotype- Tissue Expression Project) and again demonstrated relatively low expression of siglec12 in normal tissue (see Fig.7), supporting an expected advantage of lower on-target toxicities associated with systemic exposure to siglec12-targeting immunotherapeutics. Overall, expression profile results from public RNAseq database analysis indicate that siglec12 is an ideal target for antibody-based immunotherapeutic development and use for the treatment of siglec12-expressing AML, CML, and solid tumors. Example 2. Preparation of antibodies targeting siglec12 Antibodies specific for siglec12 were generated by immunizing Trianni transgenic mice (United States Publication Number US2013/0219535). Endogenous immunoglobulin VH, DH and JH; V^ and J^; and V^ and J^ gene segments have been replaced in these mice by the full repertoire of human counterpart gene segments. The human gene coding sequences are flanked by mouse cis-acting regulatory elements and recombination signal sequences allowing for a “normal” immune response, including isotype switching with heavy and light chain affinity maturation. The resulting human-mouse chimera antibodies contain human antibody variable domains flanked by mouse heavy and light chain immunoglobulin constant regions. These antibodies are more readily converted to fully human antibodies using standard methods to combine the human variable domains with human constant regions. Trianni animals were immunized with an antigen prepared by fusion of soluble human siglec12 extracellular (ecto) domain (S12ecto, UniProtKB Q96PQ1, amino acids 1- 481, SEQ ID NO: 171) with a mouse Fc2a fragment (SEQ ID NO: 177), the resulting fusion protein referred to herein as “S12-Fc.” Mouse Fc was used in place of commonly used human Fc to minimize generation of antibodies against the Fc region. For S12-Fc preparation, DNA encoding full length human S12ecto was codon optimized, synthesized, and inserted into plasmid vectors for transient transfection and expression in mammalian cells. Expressed fusion protein was purified using protein A/G SEPHAROSE®. After 6 weeks of immunization, serum samples from immunized mice were screened for the presence of siglec12-specific immunoglobulins. Screening was carried out by enzyme-linked immunosorbent assay (ELISA) and by fluorescence-associated cell sorting (FACS). ELISA assays utilized assay plates coated with S12-Fc target antigen for immobilization of siglec12-specific immunoglobulins from mouse serum samples, which were detected using anti-mouse light chain (kappa and lambda) secondary antibody conjugated to horseradish peroxidase (HRP). The level of bound secondary antibody was assessed by incubation with colorimetric peroxidase substrate followed by measurement of light absorbance intensity values obtained at 450 nm. For FACS analysis, serum samples were combined with U937 cells (Fc receptor blocked with 10% human serum), an AML cell line that overexpresses human siglec12. Immunoglobulin binding to siglec12 associated with these cells is a strong indicator of potential immunotherapeutic use to treat AML. Anti-mouse heavy chain fluorescent detection reagents were used to label cell surface-bound serum immunoglobulins before FACS analysis. Lymph node and spleen cells from the three best responders were harvested and fused with P3X63Ag8.653 mouse myeloma cells. After 10-14 days of selection, the resulting hybridoma library was single-cell cloned into 96-well culture plates using FACS. After 10-14 days of single cell expansion, all monoclonal hybridoma culture supernatants were screened for S12-Fc binding immunoglobulins by ELISA. Supernatant positive for S12-Fc-binding immunoglobulins was further screened by FACS for immunoglobulin binding to U937 cells. Results are shown in Table 12. The Table includes an antibody identification number (ID#) assigned to siglec12-binding antibodies associated with each hybridoma culture. For ELISA results, background adjusted mean absorbance values obtained at 450 nm are shown. For FACS analysis, Flow Fluor MFI is the mean fluorescence intensity from flow cytometry assay (background MFI=100) and flow % gated is the percentage of cells having greater than background fluorescence. Table 12. Antibody screening analysis
Many of the antibodies tested demonstrated strong association with U937 cell surfaces (flow % gated >10), including S0016, S0017, S0025, S0033, S0041, S0044, S0045, S0048, S0049, S0057, S0061, S0067, S0070, S0074, S0079, S0082, S0083, S0084, S0086, S0087, S0088, S0092, S0094, S0097, S0098, S0105, and S0106. Antibodies were isolated from mouse ascites fluid or from hybridoma culture media by protein A capture and used for further analysis according to the following examples. Example 3. Antibody characterization by siglec12 domain specificity ELISA assays were conducted to assess siglec12 domain specificity of antibodies identified during hybridoma screening described above. Antibodies binding to specific subdomains of S12ecto domain could provide certain advantages. For example, it is known that siglec12 expresses two different membrane-bound isoforms: a long form (595 aa, UniProtKB Q96PQ1-1, SEQ ID NO: 172) containing four extracellular domains plus a transmembrane and cytosoplasmic domain; and a shorter isoform (477 aa, UniProtKB Q96PQ1-2, SEQ ID NO: 173) that contains only a single extracellular domain (Vset domain) plus the same transmembrane and cytoplasmic domain. Antibodies that bind to the Vset2-C2set1 region of siglec12 (S12-V2C1) have the advantage of recognizing both long and short siglec12 isoforms. From a therapeutic standpoint, where both long and short siglec12 isoforms are expressed on a tumor cell, a S12- V2C1-binding antibody will bind a greater number of siglec12 molecules on the cell surface of a targeted cell, thus increasing the sensitivity and efficacy of the therapy. Antibodies that specifically bind to the Vset1 region of siglec12 (S12-V1) have the advantage of discriminating between the long and short siglec12 isoforms. Such antibodies may be useful for targeting tumor cells expressing only the long isoform of siglec12, while not targeting normal cells expressing the short form of siglec12. S12-V1 antibodies may also be useful from a biomarker standpoint for discriminating between long and short siglec12 isoforms. Antibodies that specifically bind to the C2 region of siglec12 (S12-C2) may be advantageous in targeting a region of the siglec12 extracellular domain that is closer to the cell surface. Binding to cancer epitopes closer to the cell surface has been shown to provide greater sensitivity with respect to cell cytotoxicity in the context of a CD3-mediated bispecific antibody (e.g., see Estey E.H. et al., Am J Hematol.2018.93:1267-91), thus S12- C2-specific antibodies may be candidates for S12-CD3 bispecific antibody development and immunotherapy. Proteins corresponding with subdomains of S12ecto domain were prepared for use as ELISA target antigens. The subdomains prepared included S12-V1 (amino acids 1-142 of UniProtKB Q96PQ1, SEQ ID NO: 174), S12-V2C1 (native leader sequence with amino acids 143-358 of UniProtKB Q96PQ1, SEQ ID NO: 175), and S12-C2 (native leader sequence with amino acids 365-481 of UniProtKB Q96PQ1, SEQ ID NO: 176) domains. The two domains Vset2 and C2set1 were combined since there is a naturally occurring inter-disulfide bond between the Vset2 and C2set1 domains of siglec12. To produce subdomain proteins, plasmids encoding each of S12-V1, S12-V2C1, and S12-C2 were prepared with additional nucleotides encoding a 6-His affinity tag (SEQ ID NO: 179), in addition to a StrepII affinity tag (aa sequence WSHPQFEK (SEQ ID NO: 180)) at subdomain C-termini for purification. Plasmids were transfected into human cell cultures for protein expression and subdomain proteins were isolated from culture media using Ni-IMAC (immobilized metal affinity chromatography) and STREP-TACTIN® SEPHAROSE® (GE Healthcare, Waukesha, WI) affinity chromatography purification. For ELISA assays, purified subdomain proteins were passively immobilized on assay plates and incubated with media from hybridoma cultures described above before detection of bound anti-subdomain antibodies using anti-mouse (heavy/light) secondary antibody conjugated to horseradish peroxidase (HRP). Levels of bound secondary antibody were assessed by applying colorimetric substrate and measuring absorbance values at 450 nm. Mean absorbance values obtained for antibodies associated with each hybridoma culture are presented in Table 13. Absorbance values greater than 0.07 indicate specific binding. Table 13. Antibody characterization analysis
Example 4. Antibody characterization by inter- specificity ELISA assays were also conducted to assess inter-siglec antibody specificity for other CD33-related siglecs. Siglec12 is a member of the CD33-related siglec family. From the family, human siglec7 and siglec9 have the greatest sequence identity to human siglec12. Specificity for siglec12 over other CD33-related siglecs by candidate antibodies is a desirable property for reducing potential toxicity associated with off-target binding. Fusion proteins of human siglec7 or siglec9 ectodomains fused with human Fc domains (R&D Systems, Minneapolis, MN) were immobilized on assay plates and incubated with media from hybridoma cultures described above before detection of bound antibodies using anti-mouse (heavy/light) secondary antibody conjugated to HRP. Levels of bound secondary antibody were assessed by applying colorimetric substrate and measuring absorbance values at 450 nm. Mean absorbance values obtained for antibodies associated with each hybridoma culture are presented in Table 14. Table 14. Antibody inter-siglec specificity analysis
Of the antibodies with strong association with U937 cell surfaces (flow % gated > 10), most demonstrated lack of specificity for siglec7 and siglec9 (A450 >0.09), including S0016, S0017, S0025, S0033, S0041, S0044, S0045, S0048, S0049, S0057, S0061, S0067, S0070, S0074, S0079, S0082, S0083, S0086, S0087, S0088, S0092, S0094, S0097, S0105, and S0106. Example 5. Siglec12-dependent internalization Internalization of siglec12-bound anti-siglec12 antibodies was assessed in U937 AML cells using anti-siglec12 antibodies described in Example 2. U937 AML cells were cultured and duplicate wells were incubated with or without anti-siglec12 antibodies (described in Example 2) or anti-CD33 antibody (P67.6, BioLegend, San Diego, CA) on ice for 1 hr. Cells were washed and resuspended in 800 μL ice cold complete media (10% FBS, RPMI) before plating 100 μL per well in 96-well plates. Cells were then incubated at 37°C and 5% CO 2 to promote internalization. Internalization was halted at 15, 30, 45, 60, 90, and 120 minute time points by placement on ice before cells were harvested and washed with cold PBSB (PBS+0.1% BSA). Harvested cells were then incubated with phycoerythrin (PE)- conjugated goat anti-mouse antibody (1:400, Bethyl Laboratories, Montgomery, TX) for 30 min on ice. Cells were again washed with PBSB and analyzed by flow cytometry using a GUAVA® EASYCYTE™ Plus flow cytometer (MilliporeSigma, Burlington, MA) to assess percent surface expression and percent internalization of antibody-associated siglec12 and CD33 at the time points evaluated. A comparison of average percent surface expression over time between siglec12 (labeled using S0057 antibody) and CD33 is shown in Table 15. Table 15. Percent surface expression over time The results demonstrate similar internalization levels over time between the two antibody-associated markers. A comparison of percent siglec12 internalization observed at 2 hours with different anti-siglec12 antibodies is presented in Table 16. Table 16. Percent internalization The results show internalization activity with each antibody tested, with highest activity among antibodies S0042, S0057, and S0114. Example 6. Siglec12 intracellular trafficking Siglec12-antibody complexes were assessed for intracellular trafficking to acidified cell compartments (e.g., the lysome) using acid sensitive dyes. Trafficking of internalized antibody-target complexes to acidified cellular compartments is desirable for some therapeutic antibody applications, including some utilizing antibody-drug conjugates (ADCs). Such trafficking may facilitate ADC drug release and/or activation. ZENON® labeling reagent (Thermo Fisher Scientific, Waltham, MA) includes an anti-mouse Fab fragment labeled with a pH sensitive dye that increases green fluorescence upon acidification. S0057 anti-siglec12 antibody was pre-incubated with 750 nM ZENON® labeling reagent for 15 min in PBSB.50 nM P67.6 (anti-CD33) was similarly prepared and used for comparison (as internalized CD33-antibody complexes are known to traffic to lysosomes). Resulting complexes were added to separate wells containing U937 cells and incubated for 0, 4, or 24 hours at 37°C at 5% CO 2 . Green fluorescence was measured at each time point using a GUAVA® EASYCYTE™ Plus flow cytometer. Resulting mean fluorescence intensity (MFI) values are presented in Table 17. Table 17. Acidification analysis results Acidification was comparable between siglec12-bound and CD33-bound complexes validating the potential of siglec12-specific antibody targeting as an option for ADC therapeutics. Example 7. ADC cytotoxicity on AML cells Anti-siglec12 antibodies were assessed for utility in delivering cytotoxic payloads to cells.1 mg S0057 was used to prepare a 3 mg/mL antibody solution in PBS (pH 7.8)/30% propylene glycol and combined with 40 nmols of N-succinimidyl ester activated MYLOTARG® linker/payload (Levena Biopharma, San Diego, CA). MYLOTARG® has an acid-sensitive linker with a calicheamicin payload. The antibody solution was incubated overnight at room temperature under gentle shaking. Resulting S0057-ADC was purified from unconjugated linker/payload by buffer exchange and size-exclusion chromatography. U937 AML cells were plated in a 96-well plate and incubated in complete media (10% FBS, RPMI + 100 μg/mL human IgG) with S0057-ADC titrated from 100 to 0.1 nM. Cells were incubated for 3 days at 37°C and 5% CO 2 , after which average percent cell viability was determined using GUAVA® VIACOUNT® reagent (Luminex, Austin, TX) and GUAVA® EASYCYTE™ Plus flow cytometer. Average percent viability values with each antibody concentration tested are presented in Table 18. Table 18. Average percent cell viability S0057-ADC concentrations as low as 6.25 nM were capable of reducing cell viability by nearly half. Example 8. 2 detection in AML bone marrow Bone marrow aspirate from patients with AML or acute promyelocytic leukemia (APL, negative control) was fixed with 10% formalin and used to prepare and mount 5- micron thick sections. Antigen retrieval was performed by incubating sections in antigen retrieval buffer (pH 9) for 40 minutes. Sections were treated with blocking buffer with 10% normal horse serum and incubated with S0102 anti-Siglec12 antibody (1:150). Bound antibodies were detected using peroxidase-conjugated secondary antibodies and development with 3,3'-diaminobenzidine (DAB) chromogen. Sections were counterstained with hematoxylin and eosin and visualized microscopically. Siglec12 staining of AML blasts was observed in AML patient bone marrow, while staining was absent in APL patient tissues. Example 9. Siglec12 specificity evaluation To verify anti-siglec12 antibody specificity for native siglec12 (over other siglec family members), cross-reactivity analysis was carried out using flow cytometry. Expi 293 cells were grown in 6 well plates and transfected with 2.5 mg of pcDNA3.1 expression vectors (Thermo Fisher Scientific, Waltham, MA) with inserts encoding siglec6, siglec7, siglec8, siglec9, or siglec12 proteins with C-terminal FLAG tags. The following day, Expi293 enhancers were added. On day 2 (post-transfection) cells were washed in PBSB and aliquoted into plastic V-bottomed plates at a density of 5 x 10 5 cells/well. An aliquot was also used for Western blot analysis to confirm siglec expression (using anti-FLAG antibody as described below). Plated cells were incubated with anti-siglec12 antibodies at a concentration of 500 nM or siglec-specific control antibody (all from R&D Systems, Minneapolis, MN; Siglec6-specific clone 767329; Siglec8-specific clone 837535; Siglec9-specific clone 191240) at a concentration of 12.5 μg/mL for 2 hours on ice. Cells were then washed with ice cold PBSB and incubated with PE-goat anti mouse H/L chain secondary antibody (1:400 in PBSB) for 30 minutes on ice. Cells were again washed with ice cold PBSB and analyzed for surface staining based on fluorescence intensity determined by GUAVA® EASYCYTE™ Plus flow cytometer. Resulting MFI values are presented in Table 19. Table 19. Surface staining results All anit-siglec12 antibodies tested bound to siglec12-expressing cells and demonstrated little to no binding of other siglec proteins. For FLAG-specific Western blot analysis, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and run on 4-12% NUPAGE™ gel (Thermo Fisher Scientific, Waltham, MA). Proteins were transferred onto PVDF membranes and blocked with 3% BSA in PBS. Anti-FLAG primary antibody (anti-FLAG OTI4C5, Origene Biotechnology, Rockville, MD) was diluted 1:2000 in PBS with 0.05% TWEEN® 20 and 0.3% BSA and used to treat PVDF membranes overnight at 4°C. Membranes were washed and incubated with alkaline phosphatase (AP)-labeled goat anti mouse H/L chain secondary antibody for 30 minutes at room temperature. Membranes were washed again and developed with CDP-STAR® chemiluminescent detection reagent for digital imaging of labeled bands. Results confirmed expression of siglec proteins. Example 10. S0057 epitope evaluation An antibody binding competition assay was carried out to confirm S0057 epitope and assess epitope overlap with other anti-siglec12 antibodies. A fusion protein of S12 V2C1 (SEQ ID NO: 175) and mouse Fc region fragment m2cFc (SEQ ID NO: 178) was prepared and used to coat 96-well plates in PBS overnight at 4°C. Plates were then blocked with 3% BSA/PBS for one hour before addition of biotinylated S0057 (15 nM) and a titration of unlabeled antibody (200 nM to 6.2 nM of S0057, S0023, S0102, or S0042). Plates were incubated for two hours and washed before incubation with streptavidin-Alkaline phosphatase conjugate (1:5000 in PBS/TWEEN® 20 with 0.3% BSA) for 30 min. Plates were again washed and incubated with CDP-STAR® chemiluminescence substrate before chemiluminescence analysis using a BioTek CYTATION™ 5 instrument (Winooski, VT). Mean luminescence values obtained are presented in Table 20. Table 20. Mean luminescence values V2C1-specific antibodies S0023 and S0102 (see Example 3) did not effectively compete with S0057, thus indicating that the S0057 epitope most likely does non-overlap with S0023 or S0102 epitopes. The unlabeled S0057 demonstrated effective competition as a positive control and S0042 (V1-binding negative control) did not compete for binding. Example 11. S0057 binding affinity Siglec12 binding kinetics were assessed for S0057 whole antibody and corresponding Fab fragment (obtained by whole antibody digestion). A fusion protein of S12 V2C1 (SEQ ID NO: 175) and mouse Fc region fragment m2cFc (SEQ ID NO: 178) was prepared and used to coat 96-well plates in PBS overnight at 4°C. Plates were then blocked with 3% BSA/PBS for one hour before addition of S0057 Fab fragment or S0057 whole antibody at titrations from 3 μM to 50 pM (in 0.3% BSA/PBS) using 3-fold dilutions. Plates were then incubated for 2.5 hours at room temperature under gentle shaking. Plates were washed and incubated with secondary antibody [1:10,000 goat anti-mouse Kappa chain HRP conjugate (Bethyl Laboratories, Montgomery, TX) in 0.3% BSA/PBS/Triton X-100] for 30 min at room temperature. Plates were again washed before addition of colorimetric peroxidase substrate, quenching, and absorbance measurement at 450 nm. Absorbance values were used to determine equilibrium dissociation constant (Kd) values for Fab and whole antibody formats. S0057 Fab fragment Kd was determined to be 4.8 nM and whole antibody Kd to be 2.8 nM.
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